Recently, we developed polyethyleneglycol (PEG)-modified liposomes (Bubble liposomes; BLs) entrapping ultrasound (US) gas and reported that the combination of BL and US exposure was an effective tool for the delivery of pDNA directly into skeletal muscles of an ischemic hindlimb model with local injection. To achieve gene delivery to deeper tissues, we attempted to prepare novel Bubble liposomes which were able to be loaded with pDNA and useful for systemic injection. We prepared BLs using cationic lipid and analyzed the interaction with the BLs and pDNA using flow cytometry. The solution of pDNA-loaded BLs (p-BLs) was further injected into the tail vein of hindlimb ischemia model mice and transdermal US exposure was applied to ischemic hindlimb. The effects of transfection on angiogenic factors were investigated by real-time PCR. Blood flow was determined using a laser Doppler blood flow meter. The interaction with BLs and pDNA increased by the presence of DOTAP and short PEG chains and resulted in increased stability of pDNA in the serum. Transfection with pDNA encoding the bFGF gene using p-BLs and US induced various angiogenic factors and improved the blood flow. The gene delivery system into the ischemic hindlimb using the combination of p-BLs and US exposure could be an effective tool for angiogenic gene therapy via systemic injection.

Liposomes have been explored as potential drug and gene-delivery particles. In recent years, tumor-targeted liposomes have been developed to improve the efficacy of antitumor treatment. The C16Y peptide is a modified C16 peptide, which is derived from the laminin γ1 chain, and binds to integrins α(v)β3 and α5β1 on endothelial cells. In this study, we prepared integrin-targeted C16Y peptide-modified liposomes (C16Y-L) to enhance the intracellular uptake of drugs and genes specifically into tumor tissues. The selectivity of C16Y-L for endothelial cells and cancer cells, which strongly express integrins α(v)β3 and α5β1, was assessed by flow cytometry and confocal microscopy. The cellular uptake of C16Y-L by both cell types was higher than uptake of the un-labeled and scramble peptide-modified liposomes. Next, to ascertain the involvement of receptor-mediated endocytosis in the process, these cells were treated with C16Y-L for 1h at 37°C or at 4°C. We found that uptake was also dependent on the temperature. Moreover, to evaluate whether the uptake depended on an integrin-ligand interaction, we examined the inhibition of C16Y-L uptake using recombinant integrin αVβ3 and found that the cellular uptake of C16Y-L treated with αVβ3 integrin decreased. This result suggests that C16Y-L can selectively target cells that highly express integrin αVβ3. Thus, the modification of the C16Y peptide on a Drug Delivery System (DDS) carrier may be a feasible approach for drug or gene delivery into tumors.

Recently, we reported the accelerated gene transfection efficiency of laminin-derived AG73- peptide-labeled polyethyleneglycol-modified liposomes (AG73-PEG liposomes) and cell penetrating TAT-peptide labeled PEG liposomes using PEG-modified liposomes, which trap echo-contrast gas,"Bubble liposomes"(BLs), and ultrasound (US) exposure. BLs and US exposure were reported to enhance the endosomal escape of AG73-PEG liposomes, thereby leading to increased gene expression. However, the mechanism behind the effect of BLs and US exposure on endosomes is not well understood. US exposure was reported to induce an influx of calcium ions (Ca²⁺) by enhancing permeability of the cell membrane. Therefore, we examined the effect of Ca²⁺ on the endosomal escape and transfection efficiency of AG73-PEG liposomes, which were previously enhanced by BLs and US exposure. For cells treated with EGTA, the endosomal escape and gene expression of AG73-PEG liposomes were not enhanced by BLs and US exposure. Similarly, transfection efficiency of the AG73-PEG liposomes in ATP-depleted cells was not enhanced. Our results suggest that Ca²⁺ and ATP are necessary for the enhanced endosomal escape and gene expression of AG73-PEG liposomes by BLs and US exposure. These findings may contribute to the development of useful techniques to improve endosomal escape and achieve efficient gene transfection.

We have previously reported that the transfection efficiency of laminin-derived AG73-peptide labeled polyethyleneglycol-modified liposomes (AG73-PEG liposomes) was enhanced by echo-contrast gas entrapping PEG liposomes (Bubble liposomes, BLs) and ultrasound (US) exposure by improving endosomal escape. However, it has not been well understood whether BLs and US exposure can enhance the transfection efficiency of other carriers except AG73-PEG liposomes. In this study, to evaluate whether BLs and US exposure can be generally applied to gene delivery carriers, we focused on folate as a model ligand and examined whether BLs and US exposure could enhance the transfection efficiency of folate-PEG liposomes. Folate-PEG liposomes could internalize into cells efficiently, whereas they could not deliver genes into cytosol from endosomes sufficiently. BLs and US exposure could enhance the transfection efficiency of folate-PEG liposomes compared with folate-PEG liposomes alone without their direct induction into cells. These results suggested that BLs and US exposure could enhance the transfection efficiency of folate-PEG liposomes in the same manner as AG73-PEG liposomes. Thus, BLs and US exposure may be a promising tool to achieve efficient gene transfection into various gene carriers in general.

Flow coefficients v_{n} for n=2, 3, 4, characterizing the anisotropic collective flow in Au+Au collisions at sqrt[s_{NN}]=200  GeV, are measured relative to event planes Ψ_{n}, determined at large rapidity. We report v_{n} as a function of transverse momentum and collision centrality, and study the correlations among the event planes of different order n. The v_{n} are well described by hydrodynamic models which employ a Glauber Monte Carlo initial state geometry with fluctuations, providing additional constraining power on the interplay between initial conditions and the effects of viscosity as the system evolves. This new constraint can serve to improve the precision of the extracted shear viscosity to entropy density ratio η/s.

We clarified whether actin cytoskeleton is involved in the macrophage apoptosis induced by cationic liposomes composed of stearylamine (SA-liposomes). Externalization of phosphatidylserine induced by SA-liposomes was suppressed by cytochalasin D, a specific inhibitor of polymerization of F-actin. Furthermore, activation of PKCδ and reactive oxygen species (ROS) generation, which could be involved in the macrophage apoptosis, were inhibited by cytochalasin D. Microscopical observation revealed the co-localization of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-labeled SA-liposomes and fluorescein-labeled phalloidin, which specifically binds to F-actin, and this co-localization was also inhibited by cytochalasin D. Co-localization of SA-liposomes and F-actin was also inhibited by the pre-treatment of cells with chondroitinase ABC. These findings could be the first observation concerning the contribution of the proteoglycan-actin cytoskeleton-ROS generation pathway to apoptosis induced by SA-liposomes in macrophages.

Recently, we developed novel polyethyleneglycol (PEG)-modified liposomes (Bubble liposomes; BLs) entrapping an ultrasound (US) imaging gas and reported that the combination of BLs and US was useful for the delivery of siRNA directly into the cytoplasm. However, the results were obtained using a mixture of BLs and naked siRNA. With systemic injections, it is important to control the biodistribution of both BLs and siRNA. In addition, the delivery of siRNA is affected by nuclease degradation after intravenous administration. In this study, we prepared novel siRNA-loaded BLs (si-BLs) using a cationic lipid, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). We demonstrated that siRNA could be loaded onto BLs containing DOTAP and that siRNA-loaded BLs were stable in serum. A specific gene-silencing effect was also achieved by transfection with si-BLs. Thus, the combination of si-BLs with US exposure can be used for delivery of siRNA to a specific tissue via systemic injection.

Back-to-back hadron pair yields in d+Au and p+p collisions at sqrt[s_{NN}]=200  GeV were measured with the PHENIX detector at the Relativistic Heavy Ion Collider. Rapidity separated hadron pairs were detected with the trigger hadron at pseudorapidity |η|<0.35 and the associated hadron at forward rapidity (deuteron direction, 3.0<η<3.8). Pairs were also detected with both hadrons measured at forward rapidity; in this case, the yield of back-to-back hadron pairs in d+Au collisions with small impact parameters is observed to be suppressed by a factor of 10 relative to p+p collisions. The kinematics of these pairs is expected to probe partons in the Au nucleus with a low fraction x of the nucleon momenta, where the gluon densities rise sharply. The observed suppression as a function of nuclear thickness, p_{T}, and η points to cold nuclear matter effects arising at high parton densities.

We present measurements of J/ψ yields in d+Au collisions at sqrt[s_{NN}]=200  GeV recorded by the PHENIX experiment and compare them with yields in p+p collisions at the same energy per nucleon-nucleon collision. The measurements cover a large kinematic range in J/ψ rapidity (-2.2<y<2.4) with high statistical precision and are compared with two theoretical models: one with nuclear shadowing combined with final state breakup and one with coherent gluon saturation effects. In order to remove model dependent systematic uncertainties we also compare the data to a simple geometric model. The forward rapidity data are inconsistent with nuclear modifications that are linear or exponential in the density weighted longitudinal thickness, such as those from the final state breakup of the bound state.

Oral squamous cell carcinoma is the most common type of head and neck cancer. Recently, efficient, easy, and minimally invasive gene delivery methods are expected to be developed as cancer gene therapies. However, the optimal method for delivering therapeutic genes into oral tissue for cancer treatment has not been elucidated. Therefore, we hypothesized that the tongue is a good target tissue for gene delivery with bubble liposomes and ultrasound. To assess this, we attempted to deliver a mixture of plasmid DNA encoding a luciferase or enhanced green fluorescent protein, and bubble liposomes into murine tongue with or without ultrasound exposure. The ultrasound conditions were 1MHz, 2W/cm(2), 60s, and duty cycle: 50%. The time-course of gene expression in the tongue was investigated with a luciferase assay and fluorescent microscopy. Luciferase expression was significantly increased in tongue transfected using bubble liposomes and ultrasound compared with that of the tongue untreated with ultrasound, and this high level of luciferase activity was maintained for 2 weeks. From these results, bubble liposomes can be used in combination with ultrasound to efficiently deliver plasmid DNA into the tongue in vivo. This technique is a highly promising approach for gene delivery into oral tissue.